The present invention relates to a method for detection and interpretation of disease related mutations through the combination of haploid gene transfer with functional, immunological or other analysis of the gene product.
Detection of disease-causing mutations is a complex and challenging task in medical and veterinary genetics and research. Unfortunately, loss-of-function mutations, including partial loss-of-function mutation, or gain-of-function mutations, including alteration of function and dominant negative mutations, causing inherited genetic diseases are a common problem for humans and other animals. Complete and effective detection of these mutations presents enormous possibilities as a diagnostic, preventative, or research tool.
Currently genomic sequencing of peripheral blood DNA is widely used for identification of genetic mutations associated with various diseases. In particular, it may be used to detect mutations in individuals for inherited genetic diseases. For example, Myriad Genetics, Inc. (Salt Lake City, Utah) has developed a genetic test for detection of loss-of-function mutations in BRCA1 and BRCA2, genes which have been linked to breast cancer. This test sequences all coding exons of BRCA1 and BRCA2, making it labor-intensive and costly. In addition, it cannot detect deleted exons, inversions, mutations causing loss of transcriptional activity, etc. As a result, many mutations in these two genes cannot be meaningfully detected by genomic sequencing. Table 1 displays the types and frequencies of mutations found in the BRCA1 and BRCA2 genes. Furthermore, when diploid cells that are heterozygous for a loss-of-function or a gain-of-function mutation are tested, the wild type allele can often mask the mutant allele. As a result, this test may not be accurate in detecting single mutant alleles. The usefulness of this and other such tests to the medical and veterinary professions and research scientists is therefore limited by their diagnostic shortcomings and prohibitive costs.
The Protein Truncation Test (PTT) is another diagnostic test available for the detection of loss-of-function alleles, which involves in vitro transcription and translation of the gene of interest, followed by gel electrophoretic analysis. This test is designed to detect mutations that produce a truncated protein. While this test provides an efficient means of detecting nonsense mutations, it is of no real use for detection of many other common mutations, such as frameshift, missense, inversions, and other mutations that have no detectable effect on the size of the transcribed protein.
Microarrays present another means of detecting mutations. In these assays thousands of specific oligonucleotides complementary to all known base substitutions, insertions and deletions for a gene of interest are bound to glass slides. Fluorescently labeled PCR-amplified fragments from the gene of interest are then hybridized to the microarray and binding to a particular oligonucleotide is detected. Microarrays have high up-front costs and are also not accurate at detecting heterozygous mutations. They are further limited to detection of mutations represented in the oligonucleotides.
A number of indirect methods for molecular detection of mutations exist. These include single-strand conformation polymorphism, denaturing gradient gel electrophoresis, denaturing high-performance liquid chromatography and other electrophoretic or enzymatic-based methods. Each of these methods is limited in the types of mutations it can detect and in its ability to detect heterozygous mutations in general.
To overcome the difficulty in the detection of heterozygote genotypes for inherited genetic disorders, Yan., xe2x80x9cConversion of diploidy to haploidyxe2x80x9d, Nature 403: 723-724 (February, 2000) (Yan (1)), Yan et al., xe2x80x9cGenetic testing-Present and Futurexe2x80x9d, Science 298: 1890-1891 (September, 2000) (Yan (2)), and Zoghbi et al., xe2x80x9cAssignment of Autosomal Dominant Spinocerebellar Ataxia (SCA1) Centromeric to the HLA Region on the Short Arm of Chromosome 6, Using Multilocus Linkage Analysisxe2x80x9d, Am. J. Hum. Genet. 44: 255-263 (1989) have all proposed a method of genetic testing using somatic cell hybrids haploid for a chromosome of interest. This method manipulates the two copies (alleles) of a gene of interest from a donor cell by separating the two chromosomes so that each can be analyzed individually. Detection of heterozygous mutations by these methods is improved in such cells because the wild type allele has been eliminated and cannot mask the mutated allele. However, the method described requires extremely labor intensive and impractical techniques for the isolation and segregation of haploid hybrids bearing the desired chromosome in a haploid state. Further, while the nucleic acid analysis of the haploid cells would facilitate detection of exon deletions, inversions, and transcriptional defects, the approach does not offer a significant advantage over traditional methods. Yan (2) admit that xe2x80x9c[i]t is important to note that Conversion [the Yan et al. approach] is not a substitute for the [traditional] detection methods described above, but rather is an adjunct that provides improved nucleic acid templates that can maximize the sensitivity of conventional methodsxe2x80x9d, Science 289, p.1892. Yan (2) further admit that xe2x80x9c[d]isadvantages of the Conversion [Yan et al.] approach include the increased time and expense associated with the hybrid generation and screening processxe2x80x9d, Science 289, p.1892. Thus, while the proposed method offers an improvement over the conventional screening methods, reliance on the conventional methods is not abolished and the improvement in detection is slight, especially in light of the dramatic increases in time and expense associated with the method.
Several other methods of transferring one or multiple chromosomes to a host cell have been previously described (U.S. Pat. No. 4,806,476; WO 00/34436; U.S. Pat. No. 6,077,697). This method, microcell-mediated chromosome transfer (MMCT) was first described by Fournier and Ruddle for the transfer of murine chromosomes from one cell to another (PNAS 74: 319-323 (1977)) and by McNeill and Brown for the transfer of single human chromosomes from one cell to another (PNAS 77:5394-5398 1980). MMCT describes a way of generating microcells, by prolonged colcemid and cytochalsin B treatment of donor cells, which contain one or more chromosomes or chromosomal fragments from donor cells, and fusing them using polyethylene glycol (PEG) with target cells to generate microcell hybrids, haploid for the desired chromosome/chromosomal fragment from the donor cell (FIG. 2). While these papers presents an efficient means of generating haploid cells, they fail to describe a method employing easily obtainable donor cells. In the paper of Fournier and Ruddle, mouse embryo fibroblasts were used as donors for microcell-mediated chromosome transfer. McNeill and Brown utilized human foreskin fibroblasts as donors for human chromosome transfer.
Therefore, there is a need for a medically, veterinarily, or scientifically useful method of detecting loss-of-function mutations, including partial loss-of-function mutations, or gain-of-function mutations, including alteration of function and dominant negative mutations, in any of a variety of genes. The present invention addresses the deficiencies of the prior art by providing a method for genetic testing using easily obtainable sources of genetic material that can 1) detect many types of mutations, including nonsense, missense, frameshift, deletions, inversions, etc., 2) easily detect heterozygous and homozygous mutations, and 3) less time-consuming, labor-intensive and cheaper than known methods of genetic testing.
The present invention relates to a method for detection and interpretation of loss-of-function or gain-of-function mutations for test genes of interest. The present invention involves the process of obtaining a sample of genetic material from an individual in the form of tissue or cells, separation of the genetic material from the cells of the individuals into haploid sets by transferring the individual chromosomal entities into a population of target cells, and monitoring the target cell population for successful transfer and expression of the test genes of interest using various functional, immunological and structural assays (FIG. 1). Preferably, the test gene or genes of interest are associated with known inherited human and animal disorders.
In an embodiment of the invention, the sample of genetic material from an individual with a potential genetic abnormality is in the form of cells or tissue sample. The donor cells from the individual may be any cell type obtained from the individual. In another embodiment of the invention, the individual would provide a blood sample containing peripheral blood cells. In a further embodiment of the invention, donor cells may be lymphoblasts prepared from the individual""s blood.
The genetic material comprising the test gene or genes may be located on naked DNA, plasmid, chromosome or chromosomal fragments. In a preferred embodiment of the invention, the test gene is located on a chromosome or chromosomal fragment.
In an embodiment of the invention, the separation of genetic material from donor cells into haploid sets by transfer to a population of target cells can be accomplished using various known methods of gene transfer. In a preferred embodiment of the invention, microcell mediated cell transfer (MMCT) is used to transfer genetic material to target cells.
In a preferred embodiment of the invention, the target cells may be any cell which is capable of accepting genetic material from donor cells, retaining it as a stable entity and expressing the test gene product. In a preferred embodiment of the invention, the test gene product is expressed at detectable levels. Expression of the test gene may occur through endogenous cell machinery or through cellular and molecular manipulation of cells.
In an embodiment of the invention, the presence of the test gene or genes are monitored in the target cells. In a preferred embodiment of the invention, the test gene product is monitored in the target cells. In a most preferred embodiment of the invention, the test protein is monitored. Immunofluorescence may be employed to detect test protein of interest.
In an embodiment of the invention, presence of the test gene or genes is detected by fluorescence in situ hybridization (FISH) or chromosomal painting. In yet another embodiment of the invention, the presence of the test gene is detected by fluorescent-activated cell sorting (FACS) analysis.
In another embodiment of the invention, the test gene or genes are detected though the use of a relevant functional assay for test protein function. This assay is designed based on knowledge of the cellular, immunological, molecular, biochemical, physiological, genetic, structural characteristics of the test gene product or products of interest. It takes into account all relevant functional information to design an appropriate functional assay. Assays which may be employed include, but are limited to, immunofluorescence, FACS, two-hybrid inhibition assay, ion channel activity, mismatch repair assay, and endocytic uptake of labeled LDL (low density lipoprotein).
In another embodiment of the invention, the presence of the test gene is monitored through the presence of a closely linked gene. The target cells may be monitored for either presence of linked gene or gene product, by fluorescence in situ hybridization (FISH), chromosomal painting, or fluorescent-activated cell sorting (FACS) analysis. In a preferred embodiment of the invention, known surface protein markers from specific chromosomes shared by the test gene may be used as the closely linked gene. The use of a relevant functional assay may also be employed to detect the presence of a closely linked gene and its gene products.
In another embodiment of the invention, the genotype of the donor individual may be determined by evaluating the ratio of the number of cells expressing the wild type gene product to the number of cells expressing the test gene product.